Akt promotes cellular survival, tumorigenesis, and is activated in response to carcinogen exposure, yet the field is hampered by a lack of small molecules that directly inhibit Akt. Moreover, the lack of crystal structure of Akt has limited rational drug development. To circumvent these limitations, we have modelled Akt and have synthesized small molecules designed to specifically inhibit Akt. These compounds are called phosphatidylinositol ether lipid analogues (PIAs). The goals of the project are 1. to evaluate these compounds for inhibition of Akt activity, 2. establish dose responsiveness, 3. establish specificity, 4. establish mechanism of action of inhibition, 5. establish suitability of these compounds for further development as therapeutic agents. Recent studies have indicated that of 24 PIAs screened, 5 inhibit Akt within minutes at concentrations in the low micromolar range. PIAs inhibit Akt translocation and selectively kill cancer cells with high levels of Akt activation. PIAs have been screened in the NCI 60 cell line panel and have a wide spectrum of activity, killing all cell lines albeit at concentration between 1-100 micromolar. COMPARE analysis showed that activity of the PIAs correlated with levels of active Akt but not total Akt. In addition, other molecular correlates of response were identified that either positively or negatively correlate with activity of the PIAs.. In 2007, we have identified another activity of PIAs, namely activation of the stress kinase p38. Activation of p38 occurs through a novel mechanism and contributes to the cytotoxicity of PIAs. In 2008, we identified another Akt-independent activity of PIAs, namely activation of the metabolic regulator, AMP regulated kinase (AMPK). These studies were important because activation of AMPK provides an additional inhibitory stimulus to mTOR. Thus, PIAs are the only compounds that have been described to use two complementary pathways (the Akt pathway and the AMPK pathway) to inhibit mTOR. In addition to evaluation and study of PIAs, we are screening other compounds and drugs as putative Akt inhibitors. These compounds include an analogue of thalidomide and an analogue of celecoxib. Although Akt inhibition has been attributed to each of these analogues, neither compound inhibited Akt in our systems. Instead we found that these compounds work through the induction a specific type of cellular stress, namely endoplasmic reticulum stress. Perhaps the most surprising and productive analysis of putative Akt inhibitors occurred when we analyzed a series of FDA approved drugs called HIV protease inhibitors. We chose to screen these drugs because their toxicities of increased blood glucose and increased blood lipids are similar to the toxicities observed when Akt is inhibited in preclinical animal studies. In our studies we showed that the lead HIV protease inhibitor, nelfinavir,caused the death of every cell line in the NCI 60 cell line screen at concentrations that are achievable in humans. Nelfinavir caused cellular death through programmed cell death, as well as the induction of ER stress and autophagy. Despite our initial hypothesis that nelfinavir might inhibit Akt, Akt inhibition was transient in our studies and was cell line specific. Nelfinavir also caused the death of lung cancer cells when grown as tumors in mice, and induced markers of ER stress, autophagy, and apoptosis in vivo. These studies have led to a clinical trial with nelfinavir in cancer patients that is now open and accruing patients at NCI. Current preclinical studies have now identified another FDA approved drug that has true synergy when combined with nelfinavir. A clinical trial with this combination is being planned.